1. Synapse formation completes the wiring of the nervous system
Birth and differentiation of neurons
Extension of axons/axon guidance
Target recognition
Synaptic differentiation and signaling between nerve cells
Refinement of circuits and experience-dependent modifications

2. Synapse Formation in the Peripheral and Central Nervous System

3. Synapses: the basic computation units in the brain
Human brain consists of 1011 neurons that form a network with 1014 connections
The number and specificity of synaptic connection needs to be precisely controlled
Changes of synaptic connections and synaptic strength are the basis of information processing and memory formation

4. Aberrant synaptic connectivity and synaptic function lead to disease states
Loss of synapses in Alzheimer’s disease
In epilepsy excessive synapse formation and synaptic misfunction are observed
Genes associated with mental retardation and schizophrenia have synaptic functions
Paralysis after spinal cord injuries

6. Central Synapses and Neuromuscular Junctions (NMJs)
Neuron-neuron and neuron-muscle synapses develop by similar mechanisms
NMJs are larger, more accessible and simpler than central synapses therefore the molecular mechanisms of synapse formation are best understood for the NMJ

7. Structure of the neuromuscular junction
Mature NMJs consist of three cell types
Motor nerve
Muscle cell
Schwann cells
All three cell types adopt a highly specialized organization that ensures proper synaptic function

8. Nerve terminal:
- rich in synaptic vesicles
- active zones
- mitochondria
- axon are rich in neurofilaments
and contain only few vesicles

9. Muscle:
- junctional folds opposing the
active zones
- specific cytoskeleton at synapse
- strong concentration of ACh-R

10. Schwann Cells:
- thin non-myelin processes that
cover nerve terminal
- myelin sheet around the remaining
axon from exit site from the spinal
cord to the NMJ

11. Basal Lamina:
- present at synaptic and
non-synaptic regions, but specific
molecular composition at synapse
(e.g.: acetylcholinesterase in cleft)

12. vesicles
neurofilament
ACh-receptors
overlay

13. General Features of Synapse Formation
1) The pre- and post-synaptic cell organize each others organization (bi-directional signaling)
2) Synapses mature during development
widening of synaptic cleft, basal lamina
transition from multiple innervation to 1:1
3) Muscle and nerve contain components required for synaptogenesis (vesicles, transmitter, ACh-R)
“reorganization”

14. Stages of NMJ Development
- growth cone approaches
- non-specialized but functional contact
- immature specializations
- multiple innervation
- elimination of additional axons,
maturation

15. Clustering of ACh-R: A) Aggregation of existing receptors

16. Clustering of ACh-R: B) Local synthesis of receptors

17. The basal lamina directs clustering of ACh-Rs
Denervation and muscle elimination
(but preservation of muscle satellite cells
which will form new myotubes)
In the absence of nerve, ACh-Rs cluster
at the original synaptic site

18. Agrin Component of the basal lamina 400 kDa proteoglycan
Secreted from motor neuron and muscle
Neural form potently induces clustering of ACh-Rs in myotubes
Cultured muscle fiber
Cultured muscle fiber + agrin

19. Agrin signals through MuSK
agrin interacts with a MuSK/Masc on the muscle
MuSK is a receptor tyrosine kinase
MuSK activation leads to phosphorylation of
rapsyn and clustering of ACh-Rs

20. essential roles for agrin, MuSK, rapsyn
Mouse mutants confirm
essential roles for agrin, MuSK, rapsyn
Wild type
Agrin mutant
MuSK mutant
Rapsyn mutant

21. Summary of mutant phenotypes
Agrin -/-: few ACh-R clusters, overshooting of axons
MuSK -/-: no ACh-R clusters, overshooting of axons
Rapsyn -/-: no ACh-R clusters, but higher receptor levels in synaptic area, only limited overshooting
Pre-synaptic defects in all mutants, due to the lack of retrograde signals from the muscle

22. A) Aggregation of existing receptors
Agrin
MuSK
Rapsyn
B) Local synthesis of receptors
???

23. Neuregulin (ARIA) Acetylcholine receptor inducing activity
Expressed in motor neuron and in muscle
Binds and activates receptor tyrosine kinases on the muscle (erbB2, erbB3, erbB4)
Signals through MAP-kinase pathway
Leads to upregulation of ACh-R expression in sub-synaptic nuclei

25. neuregulin (+/-) heterozygous mice
Decrease in ACh-R in
neuregulin (+/-) heterozygous mice
Wild type
Heterozygote
MEPP
(miniature
excitatory
potential)

26. Clustering of ACh-R: B) Local synthesis of receptors

27. Neural activity represses ACh-R synthesis in non-synaptic areas
Paralysis
Extra-synaptic ACh-R
transcription increased
Extra-synaptic ACh-R
transcription decreased
Denervation
Electrical
Stimulation
Extra-synaptic ACh-R
transcription increased
Extra-synaptic ACh-R
transcription decreased

28. Three neural signals for the induction of postsynaptic differentiation
Agrin: aggregation of receptors in the muscle membrane
Neuregulin: by upregulation of ACh-R expression in sub-synaptic nuclei
ACh/neural activity: downregulation of ACh-R expression in extra-synaptic nuclei

29. Components of the basal lamina can organize the nerve terminal
Denervation +
Muscle elimination
Denervation
Regeneration
Regeneration

30. Laminin 11 affects presynaptic differentiation
Wild type
Lamininb2 mutant

31. Synaptic inactivity can lead to synapse elimination
pre
post
pre
post

32. Structure of excitatory synapses in the CNS
Pre-synaptic terminal:
Synaptic vesicles
Pre-synaptic cytomatrix
Active zone
Synaptic cleft:
20 nm wide, filled with
electron-dense material
(proteins and carbohydrates)
Post-synaptic compartment:
Spine structure
Dense submembrane scaffold
Neurotransmitter receptors

33. Analogies of central synapses and NMJs
Overall structural similarities
Bi-directional signaling
Clustering of neurotransmitter receptors
Synaptic vesicles have similar components
Synapse elimination during development

34. Differences between central synapses and NMJs
No basal lamina
No junctional folds but dendritic spines
Multiple innervation is common
Difference in neurotransmitters:
Excitatory synapses use glutamate
Inhibitory synapses use GABA (g-aminobutyric acid) and glycine
different neurotransmitter receptors

35. Cytoplasmic scaffolding proteins mediate
clustering of receptors in the CNS
Gephryn
clusters
glycine
receptors
PSD95
clusters
glutamate
receptors
One neuron can receive excitatory and inhibitory inputs
through different synaptic connections
Transmitter in presynaptic vesicles is matched with the
postsynaptic receptors

36. Direct trans-synaptic interactions in the CNS
neuroligin/
neurexin
cadherins

39. presynaptic differentiation in CNS neurons
Neuroligin can induce
presynaptic differentiation in CNS neurons

40. Direct trans-synaptic interactions in the CNS
neuroligin/
neurexin
cadherins

41. Future directions/problems
Many factors that mediate synaptic differentiation in the CNS are not understood
Target specificity
Regeneration after injury is very low in CNS compared to PNS resulting in paralysis
Strategies to improve re-growth of axons and specific synapse formation